NOD2 Agonism Counter-Regulates Human Type 2 T Cell Functions in Peripheral Blood Mononuclear Cell Cultures: Implications for Atopic Dermatitis

Atopic dermatitis (AD) is known as a skin disease; however, T cell immunopathology found in blood is associated with its severity. Skin Staphylococcus aureus (S. aureus) and associated host–pathogen dynamics are important to chronic T helper 2 (Th2)-dominated inflammation in AD, yet they remain poorly understood. This study sought to investigate the effects of S. aureus-derived molecules and skin alarmins on human peripheral blood mononuclear cells, specifically testing Th2-type cells, cytokines, and chemokines known to be associated with AD. We first show that six significantly elevated Th2-related chemokine biomarkers distinguish blood from adult AD patients compared to healthy controls ex vivo; in addition, TARC/CCL17, LDH, and PDGF-AA/AB correlated significantly with disease severity. We then demonstrate that these robust AD-associated biomarkers, as well as associated type 2 T cell functions, are readily reproduced from healthy blood mononuclear cells exposed to the alarmin TSLP and the S. aureus superantigen SEB in a human in vitro model, including IL-13, IL-5, and TARC secretion as well as OX-40-expressing activated memory T cells. We further show that the agonism of nucleotide-binding oligomerization domain-containing protein (NOD)2 inhibits this IL-13 secretion and memory Th2 and Tc2 cell functional activation while inducing significantly increased pSTAT3 and IL-6, both critical for Th17 cell responses. These findings identify NOD2 as a potential regulator of type 2 immune responses in humans and highlight its role as an endogenous inhibitor of pathogenic IL-13 that may open avenues for its therapeutic targeting in AD.


Introduction
Atopic dermatitis (AD) is the leading cause of disability of all skin diseases, affecting 20% of children globally [1][2][3]. Once considered a disease of childhood, AD is found prevalent, unresolving, and burdensome across the lifespan, often manifesting with severe, treatment-resistant chronic Th2 inflammation in adults [4][5][6].
Recent studies have shown that Th2 cells require epithelial signaling at sites of inflammation before achieving full effector function or reactivation [7,8]. Keratinocytes respond to skin damage and/or infection by producing "alarmin" cytokines: IL-25 (IL-17E), IL-33, and thymic stromal lymphopoietin (TSLP) [9,10]. These alarmins prime, polarize, and recruit type 2 responses; can arrest Th1/Th17 development; and induce type 2 co-stimulatory receptors, such as OX40L on dendritic cells [11]. Alarmins are thus considered end-organ checkpoints for epithelial type 2 immune responses [12]. TSLP is highly expressed in AD skin [12] and is triggered from keratinocytes in response to injury and/or to Staphylococcus aureus (S. aureus) [13]. TSLP can even act directly on primed CD4 T cells; indeed, AD patients have elevated TSLP-receptor (TSLPR + ) T cells, which correlate with disease severity [14][15][16]. Skin-derived TSLP may in fact be sufficient to support the atopic march from skin sensitization to asthma [17][18][19][20][21]; yet, since TSLP also has homeostatic roles, co-factors in the surrounding microenvironment are likely important. AD is complicated by strong environmental influences, whereby S. aureus is a key factor, colonizing~75-90% of skin lesions [22][23][24][25][26]. Emerging evidence supports an adjuvant capacity for this pathogen to impair tolerance and drive allergic sensitization and inflammation in atopic disorders [27], yet debate persists on its bystander versus causal role in AD. S. aureus can drive inflammation via numerous virulence factors, whereby superantigens are most prominently associated, especially staphylococcal enterotoxin B (SEB) [28][29][30][31][32][33]. The severity of AD is well-known to be associated with sensitization to SEB [34]. S. aureus is also known for numerous immunoevasive mechanisms that limit effective immune responses, promoting its own survival and impeding proper immunity in humans [35,36]; as such, studies are needed to clarify such contrasting immune effects related to AD.
Protective host skin immunity to S. aureus is complex but involves innate signaling and IL-17 family cytokines, critical to recruiting neutrophils to epithelia, thus limiting S. aureus growth, and protecting the host from deep tissue invasion and infection [37,38]. As a facultative intracellular anaerobe, immunity to intracellular S. aureus also requires cell-mediated Th1 (IFNγ) responses [37]. Normal human skin has abundant S. aureusspecific tissue-resident memory CD4+ T cells, producing Th17 (IL-17A) and Th1 (IFNγ) responses [39]. In diseased AD skin, such responses are either missing, ineffective, or evaded by S. aureus [37]; instead, pathogenic Th2 (IL-13) and related cellular responses central to AD consistently prevail, which may in fact facilitate survival of S. aureus. Seminal work by Biedermann (2010) demonstrated that murine DCs coactivated by S. aureus cell-wall peptidoglycan (PGN) monomers act to suppress Th2 cell priming and generate protective Th1 and Th17 responses [40]. The cognate receptor, nucleotide-binding oligomerization domain-containing protein (NOD)2, is now known to be essential for clearing S. aureus infections in mice [41,42]. NOD2 is expressed by human antigen-presenting cells (APCs), such as dendritic cells (DCs), as well as keratinocytes where agonism drives IL-17C in response to S. aureus [43]. Notably, Th2 and Th17 cells have been shown to be reciprocally regulated; yet, such missing 'effective' S. aureus Th17 responses remain understudied in AD [44]. Understanding mechanisms driving dysfunctional Th2 effector functions despite S. aureus skin burden is critical to AD patients; we thus sought to determine the effect of NOD2 agonism in human peripheral blood mononuclear cells and its downstream impact on type 2 T cell functions.
The aim of our study was to establish an in vitro model based on skin-derived factors relevant to AD that can replicate type 2 T-cell and pathogenic IL-13 cytokine functions in disease. Using this human model, we sought to investigate the immunomodulatory effects of key innate epithelial S. aureus sensor NOD2, specifically its capacity to regulate type 2 T cell AD-like pathology.

Study Design
Adult atopic dermatitis patients and healthy subjects consented in writing to the McGill Dermatitis Database and Biobank (REB 2020-5565) and appended case-control "Cross-talk" (REB 2017-2571) for clinical data and blood analyses. Eligibility criteria for patients included adult age, meeting Hanifin & Rajka diagnostic criteria, and moderate to very severe disease as per the Eczema Area and Severity Index (EASI); median 23.6, n = 15, absence of systemic therapy according to published wash-out periods, which align with larger cohorts found in clinical trials [45]. Age-matched healthy subjects were enrolled if exclusion criteria were met, including a lack of atopic disease and personal and/or family history (n = 7). Blood samples were collected from patients and healthy subjects. Physician reported clinical parameters (EASI, vIGA, BSA) were collected from participants. Clinical laboratory tests were used to obtain IgE levels from AD patients (normal, <200 kU/L).

In Vitro Peripheral Blood Mononuclear Cell (PBMC) Model
Freshly isolated PBMCs were resuspended in complete RPMI 1640 (Gibco) supplemented with 10% autologous human plasma, 1 mM sodium pyruvate (HyClone, Logan, UT, USA), 25 mM hepes, 100 µg/mL penicillin/streptomycin (HyClone), 2mM L-glutamine, and 1X non-essential amino acids. In a 24-well plate coated with collagen I from rat tail (Corning, Corning, NY, USA), 2.5 × 10 6 cells/well in 450 µL were seeded in triplicates and rested overnight at 37 • C in a 5% CO 2 incubator. To replicate the inflammatory milieu of AD skin, PBMCs were stimulated with 2 ng/mL of staphylococcal enterotoxin B (SEB) (Toxin Technologies, Sarasota, FL, USA) and 120 ng/mL of recombinant human thymic stromal lymphopoietin (TSLP) (Biolegend, San Diego, CA, USA) at Day 0. Every 2 days, complete RPMI was added to each well. On day 7, the cells were harvested and cell count for each condition was performed. In 96-well V bottom plates, 2.5 × 10 5 cells/well were plated for subsequent flow cytometry experiments.

Functional Assays
Commercial and clinical S. aureus strains and fractions tested were previously described [46,47]. For NOD2 agonism, ultrapurified S. aureus peptidoglycan (PGN-SAndi, Invivogen) was used, abbreviated as NOD2L. Cells were seeded in 96-well plates (2.5 × 10 5 cells) and stimulated as indicated. When NOD2 agonist used, cells were incubated for 1h prior to stimulation, using vehicle as a control; cells re-stimulated using Thermo Fisher Scientific T-activator CD3/CD28 Dynabeads at a 1-to-1 ratio. Cell-free supernatants were collected and stored at −20 • C until analyzed.

Multiplex Cytokine/Chemokine Array Immunoassays
Ex vivo plasma fractions and in vitro model supernatants were analyzed using Luminex xMAP technology for multiplexed quantification of 71 Human cytokines, chemokines, and growth factors. Multiplexing analysis was performed using the Assay sensitivities of these markers range from 0.14-55.8 pg/mL for the 71-plex. Individual analyte sensitivity values are available in the MILLIPLEX ® MAP. Additional (focused) analyses were performed with in vitro model supernatants using the T helper cytokine panel (13 plex) (Biolegend). Briefly, supernatants were incubated with variable size beads that captured each specific cytokine; bead-cytokine complexes were then incubated with fluorescent-labeled cytokine-specific detection antibodies. Mean fluorescence intensity (MFI) and FSC-A (beads size) were used to quantify each cytokine's concentration using flow cytometry.

Statistical Analysis
Statistical testing varied according to the experimental design and is specified in the figure legends. Briefly, when normality could not be assumed in exploratory multiplex immuno-assay, the non-parametric Mann-Whitney test was used to compare the two unpaired groups, and p values were adjusted for multiple comparisons using Holm-Šídák's test with an alpha 0.05. In sub-analyses of target cytokines with Gaussian distribution, the two groups were compared using un-paired t-tests per row, again followed by Holm-Šídák's correction for multiple comparisons. For in vitro work with matched data sets, paired ttests per row were followed by Holm-Šídák's multiple comparisons test; for non-parametric data, Wilcoxon matched pairs signed-rank test was used. For comparison of three groups defined by one factor, analyses were performed with a one-way ANOVA, followed by Dunnett's multiple comparison post hoc test. When testing multiple cytokine levels in ex vivo analyses, a one-way ANOVA was calculated for each cytokine and corrected with Dunnett's multiple comparisons test with p-values as in the post hoc test. All experiments were performed in duplicate or triplicate. A p value of ≤0.05 was considered significant.

Type 2 Inflammatory Chemokine Biomarkers Readily Distinguish Adult Atopic Dermatitis Patient Blood from Healthy Subjects
To test and validate the detection of AD biomarkers, we characterized a small cohort of adult patients meeting diagnostic and severity criteria for moderate-to-very severe AD, compared to matched healthy subjects (see Supplemental Table S1 for clinical characteristics), which align with larger cohorts found in clinical trials [45].
Using an extended multiplexed immunoassay for 71 cytokine/chemokine/growth factors in plasma, we detected significant differences in six Th2-related chemokines between AD patients and healthy subjects, as shown in Figure 1A. Five of these six chemokines are validated [48] as AD disease biomarkers: thymus and activation-regulated chemokine (TARC/CCL17), macrophage derived chemokine (MDC/CCL22), eosinophil-attracting chemokine (eotaxin-3/CCL26), cutaneous T cell attracting chemokine (C-TACK/CCL27), and monocyte chemoattractant protein-4 (MCP-4/CCL13). Interestingly, we also detected the significant elevation of I-309/CCL1, a chemoattractant for monocytes/macrophages and Th2 cells into inflammatory sites, known to be increased in AD skin lesions [49]. These chemokine biomarkers were found to correlate significantly with each other, as shown in Figure 1B and Supplemental Figure S1. TARC correlated highly significantly with EASI disease severity ( Figure 1C,D), as did the clinical laboratory test lactate dehydrogenase and platelet derived growth factor, PDGF-AA/BB, known for its effects on fibroblasts and for mediating airway inflammation and remodeling in asthma [50]. EASI correlation with other biomarkers (IgE, MDC, eotaxin-3, sCD40L, IL-9, and PDGF-A) were not significant, although trends were found ( Figure 1C). Additional significantly elevated chemokines/cytokines/growth factors (BCA-1, VEGF-A, TNFα, MCP-3, IP-10, IL-13, FGF-2, sCD40L (all, p ≤ 0.05) and IL-5, and IL-9 (both p ≤ 0.01) did not meet the threshold following adjustments for multiple comparisons. We thus found robust biomarkers in the blood of adult AD patients, including the disease severity correlate TARC, and next sought to determine upstream signals capable of inducing these factors.

Human In Vitro Model Reproduces Type 2 Inflammatory Features of AD
To study etiologic factors relevant to adult AD disease, we established a simple in vitro PBMC-derived model, as illustrated in Figure 2A. Skin tissue-derived signals may be key to the Th2 cell pathology associated with AD; in particular, there is strong evidence for both TSLP and the S. aureus-derived superantigen SEB in disease [51]. We thus exposed PBMCs from healthy subjects to TSLP and SEB in culture and tested for a core set of internationally validated [48] AD biomarkers after one week, compared with those found ex vivo in AD patients ( Figure 2B,C). We found that TARC/CCL17, MDC/CCL22, and MCP-4/CCL-13 were significantly elevated in the culture following a single exposure to TSLP/SEB, mimicking biomarkers found in AD blood ex vivo. Two of these Th2-related chemokines are produced by epithelia (CTACK/CCL27) or endothelium (eotaxin-3); as these cell types are not found in PBMCs, they were not expected to be increased. We next assessed the cytokine biomarker IL-13 in comparison to other master Th cytokines and found that TSLP/SEB significantly upregulated the Th2 cytokines IL-5, IL-9, and IL-13 in PBMCs from healthy subjects, replicating AD-like functional deviation (Figure 3). The Th2 cytokine IL-4 was very low in culture, mimicking the IL-13-dominated profile of AD [52]. No other effector Th subset cytokine (Th1, IFNγ; Th22, IL-22; or Th17, IL-17A) was significantly upregulated, nor was IL-6 or the regulatory cytokine IL-10 (data not shown). Comparable findings were generated using blood from AD patients in vitro (data not shown). Taken together, a singular exposure to TSLP/SEB is sufficient to drive PBMCs to produce AD biomarkers like TARC, known to correlate with disease severity, as well as master Th2 cytokines, without the contribution of epithelial cells nor exogenous cytokines, such as IL-4 or IL-2, nor artefactual Th1-blocking antibodies, as used in traditional in vitro Th2 models.
To examine TSLP/SEB-mediated AD-like functional Th2 deviation at the cellular level, we next utilized multiparametric flow cytometry to study immunophenotype after stimulation by staining for Th1/Th2 intracellular cytokines, as shown in Figure 4. Using PBMCs from healthy subjects, we found that TSLP/SEB led to significantly increased IL-13 + CD4 (Th2) and CD8 (Tc2) T cells ( Figure 4A), but not IFNγ + Th1 or Tc1 cells ( Figure 4B). In AD patients, Tc2 cells were not increased following in vitro stimulation. Notably, TSLP/SEB also induced significant elevation in the Th2-related co-stimulatory receptors ICOS and OX40, as well as the master Th2 transcription factor GATA3, as shown in Figure 5. TSLP/SEB thus induced Th2/Tc2 intracellular cytokine, transcription factor, and co-stimulatory receptor expression in T cells.

Functional Modulation of Pathogenic Type 2 Cells
Prior work by our group and others has demonstrated that certain S. aureus strains and cell wall products can induce immunosuppressive responses and/or Th2 inhibition [46]. Having established skin-derived factors mediating the induction of AD-related biomarkers, chemokines, and type 2 T cells, we sought to investigate counterregulatory S. aureusmediated signals known to inhibit Th2 responses [40] using our in vitro model. -related AD chemokine biomarkers tested in plasma found significantly upregulated in the adult AD patient cohort (n = 15) relative to healthy subjects (n = 7), Mann-Whitney tests per row, followed by Holm-Šídák's correction for multiple comparisons. Significant elevation for CTACK, unpaired t-test per row, followed by Holm-Šídák's correction (p < 0.000001, data not shown). (C) In vitro stimulation with TSLP/SEB induces healthy PBMCs to secrete significantly elevated AD biomarkers following one week in culture (n = 7). Paired t tests per row, followed by Holm-Šídák's multiple comparisons test. Eotaxin-3/CCL26 and CTACK/CCL27 are produced by cell-types (endothelia, epithelia) that are not found in PBMCs.  PBMCs from healthy subjects (n = 8) cultured with TSLP/SEB for one week; proportion (%) of IL-13 + (Th2/Tc2) versus IFNγ + (Th1/Tc1) CD4/CD8 T cells. Statistical significance was determined by Wilcoxon matched-pairs signed rank test. Experiments performed in duplicates or triplicates, ns = non significant.

NOD2 Agonism in PBMCs Inhibits TSLP/SEB-Induced Type 2 Cytokine Secretion
We specifically focused on NOD2 agonism as PGN is a major constituent of the cell wall and chemical PGN alterations (blocking NOD2 recognition) feature as a prominent adaptive survival mechanism for S. aureus. We have previously found that TNFα production in response to various S. aureus strains was dependent on phagosome processing [47], and NOD2 is known to co-localize with phagosomes in innate immune cells [53]; therefore, we aimed to investigate a role for NOD2 signaling (or absence thereof) role in AD.
We tested our in vitro model following priming with a NOD2 agonist and found that memory CD4 and CD8 T cell IL-13 production was significantly inhibited relative to the control TSLP/SEB alone, as shown in Figure 6A. Culture supernatants were further tested for polar T cell cytokines induced following re-stimulation at 6 hrs. We found that PBMC cultured for 6 days in the presence of TSLP/SEB alone increased IL-4, IL-5, IL-13, IL-9, IL-10, TNF, as well as IL-22 and IL-6 production, as compared to untreated cells, following mitogen restimulation. Notably, adding the NOD2L significantly reduced Th2 (IL-4, IL-13, IL-5) and IL-10 cytokines relative to TSLP/SEB alone ( Figure 6B). Interestingly, we found that NOD2 ligation significantly increased IL-6 secretion, along with smaller but significant increases in TNF and IFNγ. We also noted the lack of IL-10 induction in contrast to prior reports with cell wall extracts of S. aureus or TLR2 ligation [54,55]. We validated that similar inhibition could be replicated in PBMCs isolated from AD patients (data not shown). In summary, we found NOD2 ligation effectively counter-regulated TSLP/SEB-induced type 2 cytokines.

NOD2 Agonism in PBMCs Induces Th17-Related Transcription Factor STAT3
As NOD2 ligation significantly increased IL-6, known to be essential for Th17 development, we sought to determine if NOD2 ligation in PBMCs affected T cell transcription factors, specifically STAT3, which transduces IL-6 signaling downstream in Th17-cells. We found NOD2 agonism significantly increased phosphorylated STAT3 (pSTAT3), key to Th17 differentiation, in both CD4 and CD8 T cells in cultures at six hours following the re-stimulation ( Figure 6C, left panel) and nuclear localization of pSTAT3 but not pSTAT5; when analyzed using Imagestream ( Figure 6C, right panel), the latter was a molecule previously reported to induce Th2 differentiation downstream of TSLPR. We also found increased levels of Lck in CD4 T cells, a tyrosine kinase and CD4 co-factor known for higher expression in Th1 rather than in Th2 cells (data not shown) [56], but no significant difference in the aryl hydrocarbon receptor (AHR), STAT1, or JAK1 at this time point. In addition, we tested the effect of NOD2L on the master Th2 transcription factor GATA3 with PBMCs from AD patients. We found a trend for reduced expression of GATA3 phosphorylation in CD4 and CD8 T cells (data not shown). While we were not powered to study donor-todonor heterogeneity, we noted that PBMCs from AD patients responded less frequently to NOD2L-mediated inhibition than healthy controls (HC: 86% and AD: 67%; data not shown). These results suggest that the NOD2 inhibition of type 2 T cell from PBMCs cultured in the presence of TSLP and SEB occurs via activation of counter-regulatory signaling pathways, leading to decreased IL-13 and increased IL-6 and pSTAT3, both critical to Th2 and Th17 responses, respectively.

Discussion
The novelty of our study is two-fold. First, we demonstrate that TSLP and SEB are sufficient to drive IL-13 secretion and pathogenic effector type 2 T cell responses from healthy PBMCs in vitro, replicating AD-like cytokine, chemokine, biomarker, and T cell functions found in patients ex vivo. Second, using this model, we showed that NOD2 ligation inhibits TSLP/SEB-mediated induction of type 2 cytokines and associated Th2 and Tc2 cell functions. These findings highlight cutaneous alarmin and S. aureus signals relevant to AD inflammation and identify NOD2 as a potential therapeutic target.
While TSLP/SEB have previously been shown to increase CCL17 and IL-2 production in CD1c + DCs, their impact on T cell function was not previously tested [57]. In 2002, Soumelis et al. reported that TLSP alone can drive Th2 differentiation from naive T cells, but only in an allogeneic system with purified CD11c + DCs [12]. Watanabe et al. then demonstrated that (IL-4 + ) Th2 differentiation occurs only under artefactual blockade (anti-IL-12) of the Th1 pathway in autologous TSLP-DC-T cell cultures; without it, homeostatic memory T cells are produced [58]. Interestingly, in this study, TSLP-DCs that pulsed with SEB were shown to be activated, but the downstream effect on Th2 effector responses was not explored [58]. SEB-activation alone induces mixed (Th1,2,17), Th1-dominated responses in epithelial-T cell (skin-homing) co-cultures [51]. As such, the significance of our work is the generation of a novel, agile human model to study pathogenic type 2 T cell responses in an autologous system without foreign antigens, artefactual Th1 blockade, or costly isolation of purified DCs. In summary, a single exposure to TSLP/SEB is sufficient to drive immune hallmarks of AD, including type 2 chemokine biomarkers and pathogenic effector type 2 T cells secreting high IL-13 and IL-5 from healthy PBMCs, validated here to correlate with AD activity in adult patients with the moderate-to-severe disease [48].
After one week, TSLP/SEB-stimulated blood mononuclear cultures secreted the canonical Th2 cytokines IL-13, IL-5, and IL-9, but very limited IL-4. Interestingly, protein levels of IL-13, but not IL-4, are consistently detected and shown to be increased in AD skin across studies [52,59]. These findings suggest that the combination of skin derived TSLP/SEB is sufficient to generate canonical pathogenic AD cytokines in blood ( Figure 3B). Stimulation in vitro significantly elevated IL-13 + Th2 and Tc2 cells and increased master transcription factor GATA3 as well as the key Th2-promoting co-stimulatory receptors ICOS and OX-40, efficiently replicating cellular profiles seen in AD patients. Our findings also add to existing evidence that OX40L on TSLP-activated DCs triggers Th2 deviation in the absence of IL-12 [60]. Interestingly, several monoclonal antibodies blocking the OX40L:OX40 axis have shown efficacy in AD and are currently in advanced human trials [61]. In our model, key AD biomarkers are reproduced, including TARC/CCL17, MDC/CCL22, and MCP-4/CCL-13; of note, biomarkers derived from epithelia or the endothelium (C-TACK/CCL27 and Eotaxin-3/CCL26) were not reproduced in our model due to a lack of endothelium/epithelium.
As introduced, pathology in AD is intricately linked to S. aureus with severity associated with sensitization to SEB [34]; yet, S. aureus is also known for numerous immunoevasive mechanisms [35,36]. The contrasting immunomodulatory effects of S. aureus related to AD remain to be clarified. Healthy individuals have abundant S. aureus-specific tissueresident Th17 (IL-17A) and Th1 (IFNγ) [39], consistent with protective cutaneous immune responses to this pathogen. Similarly, prior work has shown that S. aureus cell-wall derived PGN-derived monomers can inhibit Th2 responses in favor of Th1/Th17 responses in murine models, following NOD2/TLR2 agonism [18]. In humans, NOD2 activation has been shown in dendritic cells (DCs) to promote the development of Th17 cells and in keratinocytes to induce IL-17C [62]. Th17 cell function at epithelial surfaces is critical to clearing S. aureus via neutrophil recruitment [37]. In contrast, Th2 cell responses in AD may be conducive to survival of S. aureus, thus leading to positive microbial feedback via increased skin alarmins triggered by this virulent pathogen. While TLR2-mediated signaling is well defined, there are large gaps in understanding the wide range of NOD2-mediated immune responses in humans. NOD2 is recognized for its key role at epithelial surfaces with high microbial loads, such as the gut and the skin, co-localizing with phagosomes and activating autophagy [63,64]. NOD2 is thus poised to respond to the high burden of S. aureus peptidoglycan in AD and should drive protective antibacterial innate immune responses [53,[65][66][67][68]. Given that a central facet of S. aureus virulence and manipulation of immune responses is its capacity to co-opt phagocytic programs, we postulate that AD pathobiology may involve altered phagosomal NOD2 activation by S. aureus. Thus, we sought to clarify NOD2-mediated modulation of type 2 T cell responses, more precisely whether innate NOD2 signaling may have the potential to counteract AD-like pathobiology.
We demonstrate here that the addition of a NOD2 ligand during in vitro type 2 T cell deviation restricts TSLP/SEB-mediated induction of type 2 cytokines and T cell functions, including the inhibition of the master AD cytokine IL-13, along with IL-4 and IL-5 secretion ( Figure 6). NOD2 agonism increased IL-6 and pSTAT3 in healthy PBMCs, both critical regulators of the differentiation and function of Th17 cells [69]. Our data suggest that NOD2 activates innate myeloid cells (monocyte/macrophages and DCs) in our PBMC model, driving IL-6 secretion and downstream pSTAT3 activation in T cells. Although prior reports in a murine model indicated that NOD2 synergizes with TLR signaling to boost Th1 and Th17 responses and to suppress Th2 cell responses via DC-produced IL-10 [54,55], to the best of our knowledge, we are the first to demonstrate that NOD2 signaling in human PBMCs inhibits type 2 memory T cell expansion without IL-10 or exogenous TLR ligands. Notably, recent findings have shown that high IL-6 responses to heat-killed skin bacteria by human innate lymphoid cells is also NOD2-dependent [70,71]. The clinical relevance of the Th17-Th2 counter-regulatory mechanism is also highlighted by the monogenic Hyper-IgE syndrome, whereby defective STAT3 leads to the cardinal features of AD, including eczematous lesions on skin, S. aureus infections, and type 2 immune responses with high IgE [72].
Our findings are supported by numerous human studies demonstrating NOD2-driven production of IL-6 [73][74][75][76], TNF and IL-17 [76,77] in PBMCs, and IL-17 in memory Th cells exposed to NOD2-activated human DCs [62]. Additional NOD2-mediated pathways can also contribute to the regulation of type 2 T and innate lymphoid immune responses and require further study. Synthetic NOD2 agonists have been shown to enhance antigen presentation with DCs, stimulating T cell activation and proliferation, which may influence TCR signal strength and/or metabolic reprogramming important for Th2 effector programs [11,76]. NOD2 agonism can also upregulate IFNγ [78] in activated PBMCs and in PMBC-epithelial co-cultures [79], consistent with our findings. NOD2 is known to bind the scaffold protein receptor-interacting serine/threonine-protein kinase 2 (RIPK2), driving additional innate pathways, including the production of antiviral type 1 interferons [80]. These pathways must be further investigated, along with the role for NOD2 in mediating autophagy and/or wound healing responses within epithelial barriers that are still being defined. Atopic patients are also frequently polysensitized to protease allergens, such as house dust mites (HDM), which can alternatively activate PRRs, activate alarmins, and stimulate Th2 responses [81][82][83][84], a mechanism mimicked by murine 'AD' models, eliciting clonal responses to protein allergens. These responses contrast with polyclonal T cell repertoires in AD, as modeled here with SEB superantigen; thus, NOD2-mediated regulation of clonal allergen-specific T cells may differ. Interestingly, a NOD1 ligand was shown to inhibit allergen-induced airway inflammation in mice [85]. In summary, human-centric studies will be required to dissect species-specific and contextual NOD2 mechanistic pathways and their impact on human Th2 functional responses and atopic disorders in general.
NOD1 and NOD2 polymorphisms have been identified in AD [86][87][88], and these receptors are interconnected, emphasizing the need for more expansive future studies [89]. S. aureus is also recognized to have its own mechanisms to impair effective host PGN responses. PGN is critical for bacterial viability and a key target for host lysozyme digestion by hydrolysis. S. aureus can increase its pathogenicity by chemically altering (O-acetylating) its PGN such that lysosomal digestion is blocked, leading to impaired host responses [90]. Recent work has shown that a lack of protective immunity to S. aureus in humans involves this PGN O-acetylation, thus limiting Th17 polarization [91]. Further studies are needed to determine if this S. aureus evasion strategy plays a role in the host-pathogen dynamics with S. aureus in AD. If so, there may be important opportunities for drug discovery, including a wide range of vaccines and treatments geared to optimize NOD2 signaling.
There are limitations to the interpretation of our findings. While the adult patients included in this study are representative of cohorts included in multinational clinical trials, larger cohorts are needed to fully understand the range of immune responses that may be found in specific ethnic populations and/or age sub-groups. In addition to validated biomarkers in AD, we found a low but significant elevation of I-309/CCL1, known to recruit Th2 cells; the role of this cytokine in AD requires further investigation. Our in vitro model replicates effector AD-like type 2 T cell responses in blood, yet this only constitutes a fraction of the full spectrum of AD immune dysfunction found in the skin, where S. aureus phagocytosis and NOD2-mediated innate immune activation take place; stromal, endothelial, and epithelial cells are not featured. Chronic tissue-specific exposure to high burden S. aureus may functionally alter in situ immunity and T cell effector programs, emphasizing the need to study NOD2 response in human tissues. T lymphocytes in barrier tissues diverge significantly from populations found in the blood and exist in states that are more adaptive to microenvironmental factors, where NOD2-driven responses may vary, especially when considering tissue-resident T cells (Trm); this will be the subject of future dedicated studies. Interestingly, the topical application of a NOD2 de-repressor has recently been shown to induce the innate clearance of S. aureus in human skin explants; associated T cell responses remain to be investigated [88]. We focused most of our in vitro work on PBMCs from healthy subjects, replicating similar findings in blood from small numbers of AD patients, but we were not powered to adequately study differences between healthy and AD groups in vitro, including responsiveness to SEB, TSLP, or NOD2L. The detailed testing of dose-dependent responses and donor variability needs future work in larger studies. Future investigation will help clarify longitudinal responses and individual cytokine kinetics, along with the impact of TCR agonism potency, including (expected) quantitative but also qualitative differences in cytokines. For example, IL-22 was induced by TSLP/SEB in vitro only upon TCR re-stimulation ( Figure 3 versus Figure 6). SEB alone has been shown to induce mixed Th2/Th1 and variable donor responses according to TCR repertoire and prior sensitization, the full characterization of which is beyond the scope of this study. S. aureus PGN O-acetylation is known to decrease inflammasome responses, yet direct impact on NOD2 has not yet been shown and, thus, is postulatory. PGN-derived signaling is complex, and its capacity to induce inflammation is context-dependent, including TLR synergy and variable chemical modifications of the PGN monomer muramyl dipeptide (MDP). In addition, the murine work with MDP found seemingly contradictory NOD2-induced Th2 responses, suggesting variation based on species, ligand sub-type, scavengers, co-factors, and/or tissue microenvironments [67]. As NOD2 signaling is intimately interconnected with TLR2 [92,93] and other PRRs and the tissue microenvironment, more detailed studies are needed with an expanded scope of ligands to elucidate how such sensors interact and signal in the context of atopic skin disease. The wide-ranging potential applications of NOD2 agonism via PGN, its monomer MDP, derivatives, and synthetic compounds constitute the basis of expansive current and global research. Future studies will help to unravel the spectrum of signaling and cellular players involved in human skin.

Conclusions
Human PBMCs exposure to TSLP/SEB in vitro can be used as a simple model for testing AD-like biomarkers and type 2 T cell responses. Our findings support emerging evidence for the role of NOD2 in regulating type 2 adaptive immune responses at the skin barrier surface in humans and highlight a novel endogenous mechanism modulating pathogenic IL-13 that may open an avenue for future translational research in AD.

Supplementary Materials:
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/biom13020369/s1, Figure S1: Correlation matrices of cytokine and chemokine profiles in blood, comparing atopic dermatitis patients versus healthy subjects' 71-plex immunoassays; Table S1: Clinical parameters for atopic dermatitis patients and healthy subjects.  Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board Adult atopic dermatitis patients and healthy subjects were consented in writing to the McGill Dermatitis Database and Biobank (REB 2020-5565) and appended case-control "Cross-talk" (REB 2017-2571) for clinical data and blood analyses.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data that support the findings of this study are included in the manuscript and supporting materials.